Over 8 million Americans suffer from peripheral arterial disease (PAD), which is typically due to atherosclerotic occlusive disease of the peripheral arteries of the limbs, causing symptoms such as ischemic ulcerations or gangrene. Biological approaches to improve limb blood flow by regenerating the endothelium seem promising for treatment of PAD. A potential cell source for endothelial regeneration is embryonic stem cells (ESCs), which are pluripotent stem cells that can differentiate into endothelial cells (ECs). However, the ability to direct differentiation into ECs remains challenging due to the low yields (typically less than 5%). ESC differentiation is known to be affected by growth factors in the conditioned medium and by the matrix upon which they are grown. With respect to determining the optimal matrix composition on ESC differentiation, I propose to utilize extracellular matrix (ECM) microarrays. This is a high-throughput approach for screening several hundreds of ECMs and ECM mixtures of defined compositions for matrix- mediated ESC differentiation into EC lineage. Therefore, the goal of this project is optimize matrix conditions that favor ESC differentiation into ECs, and then characterize the phenotype and angiogenic properties of ESC-derived ECs in vitro and in vivo. First, I will characterize the differentiation of ESC-derived ECs in vitro using a high throughput ECM microarray approach. The microarray will be comprised of micro-scale spots containing single or mixtures of various matrix proteins. For easy purification and non-invasive tracking, I will utilize ESCs transduced with a lentiviral construct that consists of an EC-specific promoter, VE-cadherin, and contains genes encoding firefly luciferase for bioluminescence imaging and red fluorescent protein for immunofluorescent tracking. Next, I will differentiate ESCs on the ECM array and select the matrix composition that optimizes EC differentiation for large-scale differentiation experiments. I will then purify ECs by fluorescently activated cell sorting and characterize the cells for EC phenotypic morphology and function. Finally, using an in vivo model of PAD (hindlimb ischemia), I will determine whether ESC-derived ECs can survive in the ischemic limb, incorporate into the vasculature, and improve function by bioluminescence tracking and laser Doppler spectroscopy flow measurement. In summary, this project will utilize innovative strategies to enhance directed differentiation of ESCs into ECs for vascular regeneration for patients suffering from PAD. The potential impact of this research to public health is a reduction in mortality and morbidity among patients suffering from PAD.